The main aims of this multi-PI proposal are to characterize the underlying mechanisms the adeno- associated virus (AAV)-hepatocyte interactions in general, and to develop number of optimized recombinant (rAAV) vectors for high-efficiency transduction of hepatocytes with minimal effective dose and with minimal immune response to capsid. In recent years, we have undertaken systematic studies to gain a better understanding of the fundamental molecular mechanisms of AAV-hepatocyte interactions and have made the following significant observations, which form the basis of the current proposal: ? Identified and site-directed mutagenesis of critical surface-exposed tyrosine, serine, threonine residues on AAV capsids, and the development of next generation of highly efficient AAV vectors. ? Demonstrated that of transient suppression of the NF-kB pathway minimized pro-inflammatory response induced by AAV-mediated transduction. ? Observed involvement of the glucocorticoid receptor (GR) pathway in the life cycle of AAV2 vectors. The following three Specific Aims will be pursued: Specific Aim 1: Studying of cross-talk between GR and NF-kB pathway during transduction of hepatocytes by optimized AAV8 vectors and possible implementation to immune response reduction. Specific Aim 2: Evaluation of safety of optimized AAV8 vectors in human hepatocytes by studying frequency of integration and possible insertional mutagenesis. Specific Aim 3: Development of capsid- and genome- optimized AAV8 vectors for efficient transduction of hepatocytes at low dose and with minimal immune response. The knowledge gained from these studies will be directly applicable in the development of the next generation of rAAV vectors for their optimal use in liver-directed gene therapy and particularly for hemophilia B.